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ORIGINAL ARTICLE
Archaea of the Miscellaneous Crenarchaeotal Groupare abundant, diverse and widespread in marinesediments
Kyoko Kubo1,5,6, Karen G Lloyd2,3,5, Jennifer F Biddle3,4, Rudolf Amann1, Andreas Teske3
and Katrin Knittel1
1Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany;2Center for Geomicrobiology, Aarhus University, Aarhus, Denmark; 3Department of Marine Sciences,University of North Carolina at Chapel Hill, Chapel Hill, NC, USA and 4College of Earth, Ocean, andEnvironment, University of Delaware, Lewes, DE, USA
Members of the highly diverse Miscellaneous Crenarchaeotal Group (MCG) are globally distributedin various marine and continental habitats. In this study, we applied a polyphasic approach(rRNA slot blot hybridization, quantitative PCR (qPCR) and catalyzed reporter deposition FISH)using newly developed probes and primers for the in situ detection and quantification of MCGcrenarchaeota in diverse types of marine sediments and microbial mats. In general, abundance ofMCG (cocci, 0.4 lm) relative to other archaea was highest (12–100%) in anoxic, low-energyenvironments characterized by deeper sulfate depletion and lower microbial respiration rates(P¼ 0.06 for slot blot and P¼ 0.05 for qPCR). When studied in high depth resolution in the White OakRiver estuary and Hydrate Ridge methane seeps, changes in MCG abundance relative to totalarchaea and MCG phylogenetic composition did not correlate with changes in sulfate reduction ormethane oxidation with depth. In addition, MCG abundance did not vary significantly (P40.1)between seep sites (with high rates of methanotrophy) and non-seep sites (with low rates ofmethanotrophy). This suggests that MCG are likely not methanotrophs. MCG crenarchaeota arehighly diverse and contain 17 subgroups, with a range of intragroup similarity of 82 to 94%. Thishigh diversity and widespread distribution in subsurface sediments indicates that this group isglobally important in sedimentary processes.The ISME Journal (2012) 6, 1949–1965; doi:10.1038/ismej.2012.37; published online 3 May 2012Subject Category: microbial ecology and functional diversity of natural habitatsKeywords: benthic archaea; marine sediments; MCG; crenarchaeota
Introduction
The archaeal phylum Crenarchaeota was originallyconsidered to comprise extremophiles. Cultivatedstrains were thermophilic or hyperthermophilicorganisms utilizing sulfur for energy metabolism(Burggraf et al., 1997). Then high numbers of pelagiccrenarchaeota were discovered in the marinewater column, indicating the presence of mesophilicor psychrophilic species (DeLong, 1992; Fuhrmanet al., 1992). Current estimates of pelagic
crenarchaeota suggest they are abundant with upto 1.3� 1028 cells in the ocean (Karner et al., 2001).Mesophilic pelagic crenarchaeota have been assignedto Marine Group I, a sister group of thermophiliccrenarchaeota. Cultivated strains are autotrophicammonium-oxidizing archaea (Konneke et al., 2005;Tourna et al., 2011). A third archaeal phylum, theThaumarchaeota, was proposed for these organismsbecause of their distinct phylogeny and physiology(Brochier-Armanet et al., 2008).
The Miscellaneous Crenarchaeotal Group (MCG),has only been defined by 16S rRNA sequences andis distinct from the cultured crenarchaeota of theThermoprotei and Thaumarchaeota (Inagaki et al.,2003). The sequences delineating the MCG weredescribed by Inagaki et al. (2003), but the same cladehad been observed before and either called theTerrestrial Miscellaneous Crenarchaeotic Group(Takai et al., 2001) or Group 1.3 (Jurgens et al.,2000). Members of the MCG have been hypothesizedto be numerically and ecologically important inoceanic deep subsurface sediments because they
Correspondence: KG Lloyd. Current address: Department ofMicrobiology, University of Tennessee, Knoxville, TN, USA.E-mail: [email protected] K Knittel, Department of Molecular Ecology, Max PlanckInstitute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen,Germany.E-mail: [email protected] authors contributed equally to this work.6Current address: Institute of Low Temperature Science HokkaidoUniversity, Sapporo, JapanReceived 22 December 2011; revised 13 March 2012; accepted 13March 2012; published online 3 May 2012
The ISME Journal (2012) 6, 1949–1965& 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12
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often dominate archaeal clone libraries using 16SrRNA genes (Parkes et al., 2005) as well as 16S rRNA(Biddle et al., 2006), which is often rapidly degradedin inactive cells and may reflect the active popula-tion (Felske et al., 1996).
Currently, the carbon and energy sources for theMCG are unknown. Biddle et al. (2006) showed thattotal archaea in MCG-rich sediments are hetero-trophic, using organic carbon derived from degrada-tion of fossil organic matter. To explain energeticand isotopic discrepancies, they hypothesized thatMCG might perform ‘dissimilatory’ methane oxida-tion. However, so far the distribution of MCGcrenarchaeota relative to methane and sulfate avail-ability in the environment remains unknown. DNAfosmids containing 16S rRNA gene sequences forMCG and functional genes have been identified, butlack information about the carbon and energysources for MCG (Meng et al., 2009; Li et al., 2012).
In this study, we examined the total diversity ofpublished MCG sequences to identify phylogeneticsubgroups of this highly diverse group and identifypotential biogeographical trends. We used this compre-hensive MCG database to design primers and probes tobe used in a polyphasic approach, including rRNA slotblot hybridization, DNA-based quantitative PCR, andCARD-FISH, to investigate the distribution of MCGin eleven different habitats. We provide a protocoloptimized for in situ visualization and enumerationof MCG cells. As a specific case study, we comparedMCG abundance and taxonomic composition withdepth in the White Oak River estuary, North Carolinaand Hydrate Ridge methane seeps.
Materials and methods
Sample descriptionsEleven sites were chosen for quantification of MCGon the basis of available biogeochemical and micro-bial diversity data (Table 1).
MCG phylogenetic analysisThe ARB/SILVA 106 non-redundant 16S rRNA data-base (Pruesse et al., 2007) was supplemented withsequences from the Parc database for a total of 4720MCG sequences (for details see SupplementaryInformation). A 50% conserved filter considering 804well-aligned columns between E. coli positions 86 and935 was applied in a neighbor-joining, RAxML, andPHYML analyses for tree reconstruction using onlysequences longer than 940 bp (1276 sequences). Partialsequences were added to the tree by parsimony criteriawithout allowing changes in the general tree topology.MCG subgroup designations were made when eitherprevious publications had defined a subgroup, orsubgroups with 450 sequences were monophyleticwith both distance and maximum likelihood methods.
Probe/primer developmentAn alignment of 615 MCG sequences from ARB/SILVA release 92 (September 2007) was used to
design primers specific for the marine-derivedMCGs (235 sequences total) using ARB. Sequences,coverage and specificity of probes and primers arelisted in Table 2 and Supplementary Table S1.
Total nucleic acidsDNA and RNA were extracted from 1–6 g of frozensediments using pH8-buffered phenol and beadbeating based on protocols described previously(Stahl et al., 1988; MacGregor et al., 1997) withslight modifications. Detailed protocols are pro-vided in the Supplementary Information.
Quantitative PCRqPCR for 16S rRNA genes was performed asdescribed previously (Lloyd et al., 2011). Totalarchaeal 16S rRNA genes were quantified usingprimers ARCH915F and ARC1059R, or ARCH806Fand ARCH915R; total bacterial using BAC340F andBAC515R; and total MCG using MCG528F andMCG732R or primers MCG410F and MCG528R(Supplementary Table S1). Standards were madefrom plasmids containing inserts of environmental16S cDNA from White Oak River estuary sediments.69Earc46 (accession no. JN605175) was used forarchaea and MCG, and 70c2Aarc103 (accession no.JN616271) was used for bacteria. Plasmids contain-ing non-target 16S rRNA sequences were used asnegative controls. R-squared values for qPCR stan-dard curves were between 0.993 and 0.999. No lowmelting point primer dimers were detected. For eachcore and extraction method, samples were diluteduntil the Cp decreased log-linearly with sampledilution, indicating the absence of inhibition effectsat 10-fold dilution. Amplification efficiencies (listedin Supplementary Table S2) were determined foreach individual environmental DNA sample. Inaddition, the qPCR products of three samples werecloned and sequenced to check for amplification ofnon-target DNAs (Supplementary Table S3).
Slot blot hybridizationApproximately 10–100 ng of RNA was blotted ontonylon membranes (MagnaCharge Membrane; GEWater & Process Tech., Trevose, PA, USA) intriplicate and hybridized with 33P-labeled oligonu-cleotides as described previously (Stahl et al., 1988).The probes and dissociation temperatures used inthis study are given in Supplementary Table S1;reference rRNA are given in the SupplementaryMethods. Hybridization intensity was measuredwith a blot imager Typhoon 9400 (GE Healthcare,Munchen, Germany) and analyzed with ImageQuantsoftware (GE Healthcare).
Catalyzed reporter deposition fluorescence in situhybridization (CARD-FISH)Sediment samples were fixed in paraformaldehyde,sonicated and filtered onto a polycarbonate filter as
MCG crenarchaeota in marine sedimentsK Kubo et al
1950
The ISME Journal
Table
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MCG crenarchaeota in marine sedimentsK Kubo et al
1951
The ISME Journal
previously described (Knittel et al., 2003). Cellswere embedded in 0.1% low melting point agarose(NuSieve GTG Agarose, Cambrex Bio Science Rock-land Inc., Rockland, ME, USA) and air-dried.Inactivation of endogenous peroxidases and per-meabilization of cell walls were done by incubatingthe filters in 0.01 M HCl with 0.15% H2O2 for 10 minat room temperature. CARD-FISH and subsequentstaining with 4’,6-diamidino-2-phenylindole (DAPI)followed a previous published protocol (Pernthaleret al., 2002). For dual CARD-FISH, the protocol wasrepeated on the same filters using a second probeand other fluorescently labeled tyramides afterinactivation of peroxidases of initial hybridiza-tion as described above. The given CARD-FISHcounts are means calculated from 10 to 150randomly chosen microscopic fields correspondingto 100–800 total DAPI-stained cells. Probe sequencesand formamide concentrations required for specifichybridization are given in Supplementary Table S1.The specificity of new MCG probes was evaluated byClone-FISH (Schramm et al., 2002).
cDNA clone librariesTotal RNA was reverse-transcribed and amplifiedwith general 16S rRNA-targeted archaeal primers,checked for DNA co-extraction, and sequenced asdescribed previously (Lloyd et al., 2011). Pintail(Ashelford et al., 2006) and Chimera Slayer (Haaset al., 2011) were implemented in Mothur v.1.19.4(Schloss et al., 2009) using the ARB/SILVA Ref106database, to remove 16S rRNA sequences that werelikely chimeric. Further chimeras were identifiedmanually in Pintail. Chimeric sequences were 10%overall. Sequences were clustered into operationaltaxonomic units (OTUs) based on 97% similarity.
Amplicon sequencingDNA from 0.24–0.27 mbsf extracted from CoreDecember 06, White Oak River estuary sediments,was amplified using tagged primers for pyro-sequencing of the V6 region, ARCH958F andARCH1048R. Primer details and amplification condi-tions (http://vamps.mbl.edu/resources/faq.php#tags)were according to those of the Josephine Bay PaulCenter (Woods Hole, MA, USA) by the InternationalCensus of Marine Microbes project. The protocol toprepare PCR amplicons from archaea was followed asdescribed in Huber et al. (2007). Data processing wasfollowed as described in Huse et al. (2007). The VAMPSpipeline was used to designate MCG amplicons aftermanual curation of the archaeal reference database.Further analysis of MCG tags was performed usingCLUSTAL alignments and OTU selection in Mothur.
Nucleotide sequence accession numbersRepresentative MCG sequences obtained from qPCRproducts and sequences from cDNA libraries haveT
able
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MCG crenarchaeota in marine sedimentsK Kubo et al
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The ISME Journal
been deposited in GenBank under accession num-bers HE584637 to HE584657 and JN605005 toJN605175. Amplicon sequencing data were depos-ited in GenBank SRA archive under accessionnumber SRX011653.
Results
MCG phylogenyMCG diversity in our 4720 sequence database wasgreat, with some 16S rRNA similarity values as lowas 76% between the most distant sequences. A totalof 17 subgroups of MCGs (MCG-1–MCG-17) weresupported in both distance and maximum like-lihood inferences. These included the previouslynamed MCG-1 through MCG-4 (Sørensen and Teske,2006), C3 (Inagaki et al., 2006) that we renamedMCG-15, and pMARA-4 (Nercessian et al., 2005)that we renamed MCG-16 for consistency (Figure 1).However, these previously named subgroups cov-ered only 21% of the MCG database. Our newsubgroups increase the coverage to 91%. A fewsubgroups from the literature were not explicitlyincluded in the 17 subgroup designations becausethey are subsets of some of the other MCGsubgroups. Subgroup PM-1 fell within the MCG-2,PM-2 fell within the MCG-4, PM-3 through PM-5 fellwithin the MCG-1, PM-6 fell within the MCG-3,
PM-7 fell within the MCG-8 in some treeingmethods and PM-8 fell within the MCG-17(Webster et al., 2006). pMARA-5 (Nercessian et al.,2005) fell within MCG-17. MBGC (Vetriani et al.,1999) fell within the MCG-8. Group 1.3b(Ochsenreiter et al., 2003) fell within the MCG-6.Groups MCG-13 through MCG-17 were stable, buttheir branching order varied between treeing meth-ods, so we presented them as a multifurcation. Ingeneral, there is high intra-subgroup diversity(Table 2), with most subgroups containing p92%intragroup similarity. Similarities ranged from 82%for MCG-15 to 94% for MCG-5a and MCG-2. MCG-5split into two adjacent groups each with 490%intragroup similarity in the maximum likelihoodtrees, so these were designated groups MCG-5aand MCG-5b. The smallest subcluster MCG-2 isrestricted to sequences retrieved from marine habi-tats, whereas all others are mixed and containsequences from marine, terrestrial and limnichabitats. For an overview of MCG habitats seeSupplementary Table S4.
Design and evaluation of MCG-specific oligonucleotideprobesThe design of new probes always aims at acompromise of good target group coverage and highspecificity. Owing to the great MCG intragroup
Table 2 Coverage and specificity of new MCG primers and probes
Minimumintragroup
similarity (%)
sequenceno.a
MCG410Fcoverage (%)
MCG493coverage (%)
MCG528F/Rcoverage (%)
MCG732Rcoverage (%)
Mismatches Mismatches Mismatches Mismatches
0 1 2þ 0 1 2þ 0 1 2þ 0 1 2þ
MCG-1 88 226 72 23 5 84 15 1 91 7 2 74 21 5MCG-2 94 24 100 0 0 90 10 0 95 5 0 0 100 0MCG-3 89 129 18 23 59 97 2 1 91 9 0 8 87 5MCG-4 91 60 84 14 2 87 11 2 91 8 1 59 41 0MCG-5a 94 51 88 12 0 95 2 3 83 17 0 0 0 100MCG-5b 91 140 30 62 8 83 13 4 94 5 1 87 12 1MCG-6 90 1160 0 87 13 67 31 2 90 9 1 0 13 87MCG-7 92 81 35 49 16 40 56 4 8 36 56 0 36 64MCG-8 87 592 75 17 8 90 8 2 91 6 3 33 43 24MCG-9 87 103 92 8 0 92 4 4 91 9 0 16 5 79MCG-10 84 113 94 5 1 96 2 2 94 5 1 14 7 79MCG-11 87 232 92 6 2 79 20 1 77 22 1 5 5 90MCG-12 92 62 0 2 98 95 2 3 97 0 3 5 86 9MCG-13 85 117 40 36 24 5 80 15 5 67 28 3 67 30MCG-14 84 161 57 38 5 0 33 67 0 62 38 1 16 83MCG-15 82 635 0 0 100 o1 o1 499 o1 0 499 0 0 100MCG-16 87 29 38 52 10 0 4 96 0 0 100 0 0 100MCG-17 84 393 0 0 100 0 0 100 o1 0 499 o1 0 499Ungrouped MCG 412 ND ND ND ND ND ND ND ND ND ND ND ND
MCG-1 to MCG-17 76 4720 38 41 21 55 20 25 61 15 25 19 39 42MCG-1 to MCG-12 77 3233 44 41 15 79 19 2 87 10 3 27 51 22
Hits in non-MCGArchaea (no. sequences)b
10 600 40 33 ND 4 174 ND 3 105 ND 2 11 ND
Hits in Bacteria (no. sequences)b 278 862 0 0 ND 0 0 ND 0 0 ND 0 0 ND
Abbreviation: ND, not determined. aFull-length and partial sequences. If sequence information was lacking at probe/primer target sites, sequenceswere excluded from the analysis. bNo. of sequences in SILVA Ref106 NR database.
MCG crenarchaeota in marine sedimentsK Kubo et al
1953
The ISME Journal
diversity it was impossible to target all members ofMCG perfectly. The deeply branching subclustersMCG-13–MCG-17, in particular, are not well coveredby the four newly developed probes and primersMCG410, MCG493, MCG528 and MCG732.In silico tests revealed a MCG-1–MCG-12 groupcoverage of 87% for MCG528, 79% for MCG493,44% for MCG410 and 27% for MCG732 (Table 2).
The two probes/primers with the highest groupcoverage, MCG493 and MCG528, are highly specifichaving only three and four outgroup hits within theArchaea and none within the Bacteria. Althoughboth probes showed bright signals in Clone-FISH,we recommend the use of probe MCG493 for in situdetection because of a much brighter signal inenvironmental samples. In slot blots, use of probe
Figure 1 RaxML phylogenetic tree based on 16S rRNA genes showing MCG crenarchaeotal subgroups MCG-1–MCG-17. Subgroupdesignations follow those of Sørensen and Teske (2006) for MCG-1 through MCG-4, Nercessian et al. (2005) for MCG-16 (formerlypMARA-4), and Inagaki et al. (2006) for MCG-15 (formerly C3). MCG-13 through MCG-17 multifurcate because branching order wasinconsistent between tree models. Sequences with no color were not assigned to a subgroup. Black bars indicate the total number ofsequences from our MCG database within that subgroup, with the highest value of 1160 for MCG-6 and lowest value of 51 for MCG-5a.White Oak River estuary cDNA sequences (97% OTUs) are labeled with red or blue bars for methane oxidation or methane productionzones, respectively. The first two numbers in the clone names are the depth of that sequence, followed by A for core July 05-1, B for July05-2, E for December 06 and G for July 08-2. Additional membership in each OTU is listed after the initial clone name. Terrestrial HotSprings Crenarchaeotal Group was used as outgroup and individual clone names are listed in Supplementary Tables S4 and S5. The web-based Interactive Tree of Life was used for tree and data set visualization (Letunic and Bork, 2011).
MCG crenarchaeota in marine sedimentsK Kubo et al
1954
The ISME Journal
MCG493 resulted in easily visualized slot blotsignals (Supplementary Figure S1).
In qPCR, all newly designed MCG-targeted pri-mers amplified the 16S rRNA fragment from MCG,and showed no amplification after 40 cycles foranother deeply branching crenarchaeotal group(MBGB), ANME-2a, ANME-2c, ANME-1b, Thermo-plasmatales, Desulfobacteraceae, Gammaproteo-bacteria, Cytophagales or Alphaprotobacteria.Amplification resulted in a single, sharp peak,although a small shoulder indicates possible smallcontribution from non-target sequences. However,sequencing of qPCR products resulted only in MCGsequences, which were also quite diverse indicatingthat multiple MCG subgroups are indeed amplifiedby the primers (Supplementary Table S3).
Quantification of MCG in several types of sedimentsMembers of the MCG were quantified by rRNAslot blot hybridization, qPCR and CARD-FISH in 11different marine seep and non-seep sediments andmicrobial mats. Except for oxic surface sedimentsfrom Svalbard and most parts of anoxic methano-trophic microbial mats from the Black Sea,MCG rRNA could be detected in all types ofhabitats (Table 3). Highest MCG rRNA amountswere found in seep sediments from station 19-2 atHydrate Ridge with 178 ng g�1 at a depth of 4–5 cm.This was accompanied by the highest totalarchaeal rRNA amount detected in the sedimentsinvestigated. Relative values of MCG calculatedas a percentage of total archaeal rRNA variedgreatly between sites (Figure 2), but was signifi-cantly higher in seep and non-seep sedimentswith sulfate penetration 40.15 mbsf (1.6%±1.2 vs28.0%±27.1; P¼ 0.06, 90% CI, based on a two-tailed heteroscedastic t-test, where values forall depths in a core were averaged so that eachcore contributed one value, reducing skewedweighting towards cores with many measurements).Samples with higher potential for exposure tooxygen such as those from hypersaline microbialmats in the Arabian Gulf, and the surface of non-advective sediments from Svalbard (o0.01 mbsf)had lower MCG/archaea ratios (3.9%±9.4) thanthe deep sulfate penetration sediments (P¼ 0.08,90% CI).
qPCR data conformed with slot blot hybridization(Table 3, Figure 2). As with total rRNA, thepercentage of 16S rRNA gene copy numbers relativeto that of all archaea was significantly higher insamples with sulfate penetration 40.15 mbsf(1.0%±0.8 vs 67.9%±54.7; P¼ 0.05, 95% CI).Likewise, samples with likely oxygen exposure werealso low (0.4%±0.3; P¼ 0.05, 95% CI). In two non-seep habitats MCG were exceptionally abundant: atODP site 1229 (99%, Peru Margin) and in the WhiteOak River estuary (98–121%). Values above 100%can be explained best by a reduced bindingefficiency or insufficient coverage of used general
archaeal primers. Absolute values of MCG abun-dance were not significantly different between seepvs non-seep sites (slot blot P¼ 0.47, qPCR P¼ 0.15,and CARDFISH P¼ 0.16).
As a third method for MCG quantification weadapted the standard CARD-FISH protocol(Pernthaler et al., 2002) for in situ detection of MCGcells. The permeabilization of the cell walls turnedout to be the most crucial for visualization of MCG.Archaeal cell walls are usually permeabilized eitherby a treatment with 10–15mg ml�1 proteinase K for1–5 min, 0.5% SDS for 10 min at room temperature orwith 60 U ml� 1achromopeptidase at 37 1C (Teira et al.,2004; Knittel and Boetius, 2009; Labrenz et al., 2010).However, none of these methods was successful forthe visualization of MCG. Instead, we used 0.01 M HClfor 10 min that resulted in bright signals. Higher HClconcentrations (4 0.1 M) did not increase the fractionof hybridized cells, but rather caused a visibledisintegration of cells.
Hot spots of MCG as identified by slot blots, qPCRor 16S rRNA gene libraries were selected for CARD-FISH. About 90% of the MCG cells were small andcoccoid (Figure 3) with a cell size of 0.4–0.5 mm.This is a comparable size to that reported for theirsister group Marine Benthic Group B (MBGB)crenarchaeota (0.2–0.4 mm) (Knittel et al., 2005). Aminor part of the MCG community had a muchlarger cell size with up to 1mm in diameter andmight reflect the observed large MCG intragroup 16SrRNA sequence diversity. In most cases MCG weredetected as single cells but they were also found inaggregates of 2–5 cells. DAPI signal of MCG wasclearly visible in most habitats except for all ODPsites investigated where DAPI staining did not workat all. To corroborate the identification of detectedMCG cells, dual hybridizations were performedwith probe MCG493 and the general archaeal probeARCH915. MCG493 signals were always co-loca-lized with ARCH915 signals (Figure 3I).
In White Oak River sediments 1.2� 108 cells cm�3
were detected at shallow depth of 0.075 mbsf(12% of total cell counts, 22% of total Archaea).In the subsurface at 0.405 mbsf, the absolute MCGabundance was comparably high but the relativefraction of MCG increased to 30% and 60% oftotal archaea. In other deep sulfate penetrationsediments, MCG were as dominant as in WhiteOak River sediments; they contributed 18–20% oftotal Archaea in Janssand intertidal sand flats,15–74% at ODP sites 1245 and 1250 at HydrateRidge, 22% at Peru Margin ODP site 1229, 100% atPeru Basin ODP site 1231 and 48% at easternequatorial Pacific ODP site 1225. The two lattersites showed very low microbial activities andlow organic matter content. In deep sediments(4.65 mbsf) from the crater center of Haakon Mosbymud volcano, a site which is strongly dominatedby MCG 16S rRNA sequences (Losekann-Behrens,Boetius & Knittel, unpublished), MCG cellsaccounted for 16% of total Archaea.
MCG crenarchaeota in marine sedimentsK Kubo et al
1955
The ISME Journal
Table
3Q
uan
tifi
cati
on
of
MC
GC
ren
arc
haeo
tain
div
erse
sed
imen
tsan
dm
icro
bia
lm
ats
Sit
eS
tati
on
/sam
ple
Dep
th(m
bsf
)rR
NA
slot
blo
th
ybri
diz
ati
on
qP
CR
FIS
H
Bacte
ria
rRN
A(n
g)a
Arc
haea
rRN
A(n
g)a
MC
GrR
NA
(ng)a
Arc
haea
gen
ecop
iesb
MC
Ggen
ecop
iesb
Tota
lcell
s(c
m�
3)
Arc
haea
(cm�
3)
MC
G(c
m�
3)
Oxic
mats
Ara
bia
nG
ulf
Abu
Dh
abi
Oxic
part
1.7
Eþ
03
2.6
Eþ
02
6.0
Eþ
01
NA
NA
NA
NA
NA
An
oxic
meth
an
otr
op
hic
mats
Bla
ck
Sea
P822
Top
part
NA
1.0
Eþ
05
ND
1.4
Eþ
10
1.3
Eþ
07
NA
NA
NA
Exte
rior,
ora
nge
NA
1.2
Eþ
04
5.1
Eþ
01
2.4
Eþ
09
6.2
Eþ
06
NA
NA
NA
Exte
rior,
bla
ck
NA
2.8
Eþ
04
ND
1.3
Eþ
10
2.4
Eþ
07
NA
NA
NA
Inte
rior
NA
3.2
Eþ
04
ND
3.1
Eþ
09
2.6
Eþ
07
NA
NA
NA
Oxic
sed
imen
tsS
valb
ard
Sm
eere
nbu
rg-f
jord
en
0.0
025
9.6
Eþ
03
7.7
Eþ
01
ND
2.0
Eþ
08
1.2
Eþ
06
NA
NA
NA
0.0
075
NA
9.7
Eþ
01
ND
1.8
Eþ
08
6.5
Eþ
05
NA
NA
NA
Meth
an
ese
ep
sed
imen
tsw
ith
sulf
ate
pen
etr
ati
ono
0.1
5m
bsf
Nyegga
272-0
2(S
OB
mat)
0.0
5N
A2.9
Eþ
02
6.4
Eþ
00
4.0
Eþ
09
1.6
Eþ
06
NA
NA
NA
Casc
ad
iaM
arg
in19-2
0.0
05
NA
1.1
Eþ
03
3.1
Eþ
01
1.4
Eþ
08
3.1
Eþ
05
NA
NA
NA
Hyd
rate
Rid
ge
0.0
15
NA
2.0
Eþ
03
1.6
Eþ
01
2.4
Eþ
08
9.8
Eþ
05
NA
NA
NA
0.0
25
NA
8.1
Eþ
03
3.4
Eþ
01
5.4
Eþ
08
1.7
Eþ
06
NA
NA
NA
0.0
35
NA
2.7
Eþ
04
8.6
Eþ
01
2.7
Eþ
09
5.1
Eþ
06
NA
NA
NA
0.0
45
NA
5.2
Eþ
04
1.8
Eþ
02
1.7
Eþ
09
6.0
Eþ
06
NA
NA
NA
0.0
55
NA
2.4
Eþ
04
7.7
Eþ
01
1.1
Eþ
09
6.9
Eþ
06
NA
NA
NA
0.0
65
NA
2.7
Eþ
03
8.4
Eþ
00
4.4
Eþ
08
3.7
Eþ
06
NA
NA
NA
0.0
75
NA
3.7
Eþ
03
1.2
Eþ
01
7.8
Eþ
08
5.1
Eþ
06
NA
NA
NA
0.0
85
NA
1.4
Eþ
04
1.9
Eþ
01
5.8
Eþ
08
1.4
Eþ
06
NA
NA
NA
0.0
95
NA
3.0
Eþ
03
ND
4.5
Eþ
08
1.6
Eþ
06
NA
NA
NA
105
0.0
05
NA
6.9
Eþ
02
ND
5.1
Eþ
05
NA
NA
NA
NA
0.0
15
NA
1.6
Eþ
03
2.2
Eþ
01
5.4
Eþ
05
NA
NA
NA
NA
0.0
25
NA
1.4
Eþ
03
1.4
Eþ
01
4.2
Eþ
05
NA
NA
NA
NA
0.0
35
NA
4.0
Eþ
02
ND
ND
ND
NA
NA
NA
0.0
45
NA
1.0
Eþ
03
9.1
Eþ
00
1.6
Eþ
06
NA
NA
NA
NA
0.0
55
NA
1.4
Eþ
03
1.8
Eþ
01
ND
ND
NA
NA
NA
0.0
65
NA
2.0
Eþ
03
3.3
Eþ
01
3.4
Eþ
05
NA
NA
NA
NA
0.0
75
NA
1.9
Eþ
03
4.0
Eþ
01
2.3
Eþ
06
NA
NA
NA
NA
0.0
95
NA
1.7
Eþ
03
2.2
Eþ
01
ND
ND
NA
NA
NA
0.1
7N
A7.0
Eþ
02
8.0
Eþ
00
ND
ND
NA
NA
NA
38
0.0
4N
A2.8
Eþ
03
4.4
Eþ
00
2.2
Eþ
08
4.5
Eþ
05
NA
NA
NA
185
0.0
05
NA
2.3
Eþ
02
1.2
Eþ
01
ND
ND
NA
NA
NA
0.0
15
NA
6.1
Eþ
02
1.0
Eþ
01
ND
ND
NA
NA
NA
0.0
45
NA
4.4
Eþ
02
4.3
Eþ
00
ND
ND
NA
NA
NA
0.1
2N
A4.9
.Eþ
03
1.4
.Eþ
01
ND
ND
NA
.NA
NA
Gu
lfof
Mexic
o156
0.0
1N
A1.1
Eþ
03
4.9
Eþ
01
3.6
Eþ
07
4.2
Eþ
05
NA
NA
NA
0.0
3N
A8.5
Eþ
02
3.0
Eþ
01
2.5
Eþ
07
4.4
Eþ
05
NA
NA
NA
0.0
5N
A6.7
Eþ
02
9.8
Eþ
00
2.9
Eþ
07
5.4
Eþ
05
NA
NA
NA
0.0
7N
A6.1
Eþ
02
2.2
Eþ
01
1.8
Eþ
07
4.1
Eþ
05
NA
.NA
NA
HM
MV
372
(Cen
ter)
4.6
5N
AN
AN
AN
AN
AN
A3.3
Eþ
08
5.3
Eþ
07
371
(Beggia
toa)
4.5
8N
AN
AN
AN
AN
AN
A3.1
Eþ
09
1.4
Eþ
08
Meth
an
ese
ep
sed
imen
tsH
MM
V336
(Pogon
op
hora
)4.1
0N
AN
AN
AN
AN
AN
A2.9
Eþ
09
7.6
Eþ
07
wit
hsu
lfate
Pen
etr
ati
on
Gu
lfof
Mexic
o87
0.0
17.3
Eþ
03
5.8
Eþ
02
5.8
Eþ
01
1.7
Eþ
07
7.9
Eþ
05
1.9
Eþ
09
1.0
Eþ
09
2.7
Eþ
07
40.1
5m
bsf
0.0
31.9
Eþ
02
6.8
Eþ
01
3.7
Eþ
00
2.8
Eþ
06
5.8
Eþ
05
NA
NA
NA
0.0
56.1
Eþ
00
1.6
Eþ
01
1.8
Eþ
00
2.9
Eþ
06
ND
c8.3
Eþ
08
2.1
Eþ
08
0.0
71.3
Eþ
02
4.4
Eþ
01
2.0
Eþ
00
2.8
Eþ
06
ND
NA
NA
NA
Hyd
rate
Rid
ge
OD
P1245D
1H
21.5
5–1.9
05.0
Eþ
00
3.2
Eþ
01
3.1
Eþ
00
NA
NA
1.2
Eþ
08
de
7.3
Eþ
06
1.1
Eþ
06
OD
P1250D
3H
723.9
0N
AN
AN
AN
AN
A1.1
Eþ
07
de
6.2
Eþ
06
4.6
Eþ
06
MCG crenarchaeota in marine sedimentsK Kubo et al
1956
The ISME Journal
Table
3(C
on
tin
ued
)
Sit
eS
tati
on
/sam
ple
Dep
th(m
bsf
)rR
NA
slot
blo
th
ybri
diz
ati
on
qP
CR
FIS
H
Bacte
ria
rRN
A(n
g)a
Arc
haea
rRN
A(n
g)a
MC
GrR
NA
(ng)a
Arc
haea
gen
ecop
iesb
MC
Ggen
ecop
iesb
Tota
lcell
s(c
m�
3)
Arc
haea
(cm�
3)
MC
G(c
m�
3)
Oth
er
sed
imen
tsw
ith
Nort
hS
ea
Jan
ssan
d0.0
25
NA
NA
NA
NA
NA
2.8
Eþ
08
1.1
Eþ
08
9.7
Eþ
06
sulf
ate
pen
etr
ati
on
0.1
54.4
Eþ
03
6.6
Eþ
01
2.2
Eþ
01
8.8
Eþ
06
5.5
Eþ
05
NA
NA
NA
40.1
5m
bsf
0.2
85
NA
NA
NA
NA
NA
4.6
Eþ
08
1.4
Eþ
08
2.7
Eþ
07
0.4
06.5
Eþ
03
2.1
Eþ
02
6.6
Eþ
01
NA
NA
NA
NA
NA
2.0
0N
AN
AN
AN
AN
A1.9
Eþ
08
6.2
Eþ
07
1.1
Eþ
07
4.9
2.2
Eþ
00
9.3
Eþ
00
1.4
Eþ
00
1.8
Eþ
08
2.8
Eþ
07
3.0
Eþ
08
8.4
Eþ
07
1.6
Eþ
07
Equ
ato
rial
Pacif
icO
DP
1225A
32H
3,
34H
3,
35H
5286–320
fN
AN
AN
AN
AN
AN
A2.6
Eþ
06
1.3
Eþ
06
Peru
Marg
inO
DP
1227A
5H
337.3
83.3
Eþ
01
1.0
Eþ
02
9.4
Eþ
00
NA
NA
NA
1.5
Eþ
07
3.3
Eþ
06
OD
P1229D
1H
1þ
1H
20.3
25þ
2.3
25
8.5
Eþ
01
1.1
Eþ
02
8.6
Eþ
01
2.2
Eþ
08
2.2
Eþ
08
NA
NA
NA
Peru
Basi
nO
DP
1231B
9H
2,
10H
2,
11H
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12H
272–100
gN
AN
AN
AN
AN
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A2.4
8Eþ
06
2.5
Eþ
06
Wh
ite
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er
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tion
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0.0
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NA
NA
NA
6.4
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3.8
1Eþ
07
NA
NA
NA
0.0
75
NA
NA
NA
5.4
9Eþ
08
3.7
9Eþ
08
1.0
Eþ
09
5.6
Eþ
08
1.2
Eþ
08
0.1
95
NA
NA
NA
3.1
6Eþ
08
4.2
5Eþ
08
NA
NA
NA
0.2
55
NA
NA
NA
5.7
0Eþ
08
8.4
6Eþ
08
NA
NA
NA
0.3
15
NA
NA
NA
4.3
1Eþ
08
4.2
3Eþ
08
1.3
Eþ
08
5.0
Eþ
07
3.0
Eþ
07
0.3
45
1.2
Eþ
01
2.4
Eþ
01
6.5
Eþ
00
NA
NA
NA
NA
NA
0.3
75
NA
NA
NA
3.8
3Eþ
08
4.0
9Eþ
08
NA
NA
NA
0.4
05
NA
NA
NA
NA
NA
5.3
Eþ
08
3.7
Eþ
08
1.1
Eþ
08
0.4
35
NA
NA
NA
4.3
7Eþ
08
5.2
8Eþ
08
NA
NA
NA
0.4
95
NA
NA
NA
4.2
3Eþ
08
3.4
9Eþ
08
NA
NA
NA
0.5
55
NA
NA
NA
3.4
9Eþ
08
3.0
4Eþ
08
NA
NA
NA
0.5
85
NA
NA
NA
5.5
3Eþ
08
6.2
5Eþ
08
NA
NA
NA
0.6
15
8.7
622932
20.0
76825
9.1
414717
3.4
9Eþ
08
4.8
7Eþ
08
NA
NA
NA
0.6
75
NA
NA
NA
2.8
1Eþ
08
3.2
0Eþ
08
NA
NA
NA
Sta
tion
H,
core
July
08-2
0.0
45
NA
NA
NA
3.6
7Eþ
07
3.1
7Eþ
07
NA
NA
NA
0.1
05
NA
NA
NA
4.9
8Eþ
07
4.9
5Eþ
07
NA
NA
NA
0.1
35
NA
NA
NA
5.7
9Eþ
07
5.4
8Eþ
07
NA
NA
NA
0.1
65
NA
NA
NA
4.5
7Eþ
07
4.7
4Eþ
07
NA
NA
NA
0.1
95
NA
NA
NA
6.2
8Eþ
07
9.6
2Eþ
07
NA
NA
NA
0.2
25
NA
NA
NA
5.1
5Eþ
07
8.6
7Eþ
07
NA
NA
NA
0.2
55
NA
NA
NA
3.6
2Eþ
07
5.3
8Eþ
07
NA
NA
NA
0.2
85
NA
NA
NA
4.8
9Eþ
07
8.7
8Eþ
07
NA
NA
NA
0.3
15
NA
NA
NA
5.5
1Eþ
07
6.6
7Eþ
07
NA
NA
NA
0.3
45
NA
NA
NA
7.4
9Eþ
07
5.3
7Eþ
07
NA
NA
NA
0.4
05
NA
NA
NA
5.6
2Eþ
07
6.0
7Eþ
07
NA
NA
NA
0.4
35
NA
NA
NA
5.9
4Eþ
07
7.3
6Eþ
07
NA
NA
NA
0.4
65
NA
NA
NA
6.9
7Eþ
07
7.5
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07
NA
NA
NA
Abbre
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.7:1
.6.
MCG crenarchaeota in marine sedimentsK Kubo et al
1957
The ISME Journal
Figure 2 Relative abundance of MCG crenarchaeota compared with total archaea as determined for different marine habitats by rRNAslot blot hybridization (black bars), qPCR (white bars), and CARD-FISH (gray bars). Colors indicate samples from anoxic methanotrophicmicrobial mats (peach) or oxic surface sediments (pink) or sediments with sulfate depletion depths of o0.15 mbsf (yellow) or 40.15 mbsf(blue). Dots indicate samples with no MCG detected with slot blot (black) or qPCR (white); other samples with no visible data were notanalyzed with that method. When a depth range is listed, values were averaged over adjacent depths. All individual measurements listedin Table 3.
Figure 3 Single cells of MCG crenarchaeota in subsurface sediments from the sulfate–methane transition zone of the White Oak Riverestuary (0.4 mbsf; a–f) and ODP site 1227 at Peru Margin (5H3, 37.38 mbsf; g–i), visualized by CARD-FISH. a and d show DAPI staining(blue); other panels show the corresponding FISH signals obtained by dual hybridization with the general archaeal probe ARCH915 (b, e,g; red) and MCG-specific probe MCG493 (c, f, h; green). i shows an overly of g and h. Arrows point to MCG cell signals. Scale bars, 5 mm.
MCG crenarchaeota in marine sedimentsK Kubo et al
1958
The ISME Journal
MCG depth distributionIn order to explore changes in MCG abundance overdepth, we examined the White Oak River estuarycores (sulfate depleted at 0.40 mbsf) and site 19-2from Hydrate Ridge (sulfate depleted at 0.07 mbsf,Figure 4). In White Oak River core Jul 08-1 qPCRsusing duplicate primer sets gave roughly equal 16SrRNA gene copies (Figures 4a and b), suggesting lowor equal primer bias. At the division between thehorizon dominated by sulfate reduction andmethane oxidation and that dominated by metha-nogenesis DNA concentrations for archaea, bacteriaor MCG did not change perceptibly. At and below0.045 mbsf, total archaeal DNA copies matched MCGDNA copies for all four primer sets. Above, at0.015 mbsf, however, MCG 16S rRNA gene copynumbers were much lower than those of archaea.Bacterial DNA copies were consistently higher thanarchaeal and MCG DNA copies with the gapnarrowing gradually with depth. Large differencesin absolute values between July 08-1 and July 08-2correspond to differences in Cp values, whereas Cp
values for standards were nearly identical, suggest-ing much lower DNA yields during extraction of July08-2. An interesting contrast to the lack of verticalzonation of MCG in White Oak River estuarinesediments was Hydrate Ridge, where quantities andactivities of MCG and total archaea peaked around0.05 mbsf near the base of the sulfate reduction zone,as shown in both qPCR and RNA slot blot data(Figure 4c). As MCG do not increase relative toarchaea, and the archaeal peak is far too large to becaused by the MCG increase alone, this peak is mostlikely due to an increase in total ANME biomasssupported either directly or indirectly by very highrates of anaerobic methane oxidation at this site(Treude et al., 2003).
Given a lack of vertical zonation in total MCGabundance in the White Oak River estuary, weinvestigated possible changes in MCG phylogeneticcomposition with depth. MCG comprised the major-ity (44–86%) of 16S rRNA cDNA sequences fromWhite Oak River estuary sediments (Figure 5). Otherarchaeal sequences included the uncultured groupsANME-1 (Hinrichs et al., 1999), Marine BenthicGroups (MBGA, B and D Vetriani et al., 1999), DeepSea Euryarchaeotal Group (DSEG; Takai et al., 2001),or Terrestrial Hot Springs Crenarchaeotic Group(THSCG Takai and Horikoshi, 1999) (SupplementaryFigure S2). MCG had more OTUs than all otherarchaea in the sediments combined (92 OTUs vs 79OTUs, respectively, at 97% similarity) and covered 13out of the 17 subgroups (Figure 1, SupplementaryFigure S1). The general composition and sequenceabundance did not change much with depth orseason (Supplementary Table S5). No clear phyloge-netic separation was observed for sequences from themethane oxidation and production zones (Figure 1,red and blue dots, respectively). No seasonality wasobserved with MCG types, as sequences from thewinter core (December 06) were distributed
Figure 4 qPCR of 16S rDNA for White Oak River estuary coresJuly 08-1 (a) and Jul 08-2 (b) and Hydrate Ridge core 19-2 (c).Primer sets used were for total archaea (ARCH915F-ARCH1059R,filled red squares, and ARCH806F-ARCH915R, open red squares),MCG (MCG528F-MCG732R, filled blue circles and MCG410F-MCG528R, open blue circles) or total bacteria (BAC340R-BAC515R, filled black diamonds). All replicate measurements ateach depth are shown. In addition, rRNA concentrations of MCG(blue x’s) and total archaea (red x’s) measured with slot blothybridization for Hydrate Ridge core 19-2 are shown in c. Sulfate(filled triangles) and methane (open squares or modeled line)concentrations for White Oak River estuary cores July 08-1 (d) andJuly 08-2 (e) and for Hydrate Ridge (f); (originally published inLloyd et al. (2011), and in Treude et al., 2003). Dashed horizontallines are the depth above which there is net anaerobic oxidationof methane (AOM) and below which there is net methaneproduction (MP; Lloyd et al., 2011).
MCG crenarchaeota in marine sedimentsK Kubo et al
1959
The ISME Journal
throughout the MCG tree along with those fromsummer cores (July 05-2, July 08-1 and July 08-2).
Amplicon pyrosequencing supported the domi-nance of MCG within the archaea with 86% of13 250 sequences (Figure 5). It is surprising thatthe cDNA clone libraries agreed so well with theamplicon pyrosequencing, as one targets RNA andthe other targets DNA, different primers were used,and the V6 amplicon library was 150 times greaterthan the biggest cDNA clone library. The onlydifference was that the amplicon data containedsequences for MBGA and the Methanobacteriales,which were presumably in lower numbers in thesediments and therefore missed in the clonelibraries. After error processing, which includesclustering that may hide some alpha diversity,1192 unique MCG sequences were detected. Ofthese, 790 were singletons and only 19 were seen100 or more times. These 19 sequences made up57% of all tags seen within the archaeal White OakRiver data set, truly a significant portion of thearchaea in this sediment. The diversity seen withinthese tags was also high. Tags represented over 10times were aligned and OTUs were calculated.Within these 99 tags, there were 99 OTUs calculatedat 99% similarity, 73 OTUs at 98% similarity and 51OTUs still remained at 97% similarity.
Quantification of total bacterial rRNAOn the basis of the size of MCG rRNA fraction, twoseep habitats (Gulf of Mexico site 87, Hydrate RidgeODP site 1245D) and three non-seep habitats(Janssand, White Oak River, Peru ODP sites 1227A
and 1229D) were selected for quantification of totalbacterial 16S rRNA. All habitats were characterizedby deep sulfate penetration. Surface sediment fromJanssand sand flat was strongly dominated bybacterial rRNA (97–98% bacterial rRNA: 2–3%archaeal rRNA) at a depth of 0–0.4 mbsf, but byarchaeal rRNA in a deeper sediment horizon at4.9 mbsf (19% bacterial rRNA: 81% archaeal rRNA).The ratio of bacterial to archaeal rRNA percentageswas comparably low for the other subsurfacesediments investigated, that is, 14%:86% at ODPsite 1245 at Hydrate Ridge, 24%:76% at ODP site1227, 45%:55% at ODP site 1229 and 34%:66% atWhite Oak River (Table 3, Figure 6).
Discussion
When looking at all MCG sequences available inpublic databases, MCG crenarchaeota appear verydiverse and widespread. MCG are present interrestrial hot springs (Barns et al., 1996), deepoceanic subsurface sediments (Parkes et al., 2005),deep terrestrial subsurface (Inagaki et al., 2003),continental shelf sediments (Vetriani et al., 1998),ancient marine sapropels (Coolen et al., 2002),petroleum-contaminated soil (Kasai et al., 2005),termite guts (Friedrich et al., 2001), mud volcanoes(Heijs et al., 2007), methane hydrate-containingmarine sediments (Inagaki et al., 2006), landfillleachate (Huang et al., 2003), anaerobic wastewaterreactors (Collins et al., 2005), sulfidic springs(Elshahed et al., 2003), brackish lakes (Hershbergeret al., 1996) and coastal salt marshes (Castro et al.,
a
b
Figure 5 Phylogenetic groupings of all 16S rRNA cDNA clones and 16S rRNA gene V6 amplicon tag sequences for White Oak Riverestuary sediments that derived from (a) methane oxidation or (b) methane production zone. Numbers at the right side of bars are the totalnumber of sequences in the library. Abbreviations and references for phylogenetic groups are listed in results section.
MCG crenarchaeota in marine sedimentsK Kubo et al
1960
The ISME Journal
2004). Only 28 out of 4720 MCG sequences wereretrieved from potentially oxic habitats (for exam-ple, database releases EU370096 and FJ560325).This broad distribution is reflected in a large MCGintragroup diversity with 16S rRNA gene similarityvalues as low as 76%, which is within the rangeproposed to define prokaryotic phyla (Yarza et al.,2010). The diversity within most of our subgroups isin the proposed cutoff of 87.7% minimum level forfamily boundaries (Yarza et al., 2010). It is possiblethat this evolutionary diversity is also reflected in ahigh degree of metabolic diversity.
MCG crenarchaeota constituted a major part of archaeain sediments with low respiration rates and deepsulfate penetrationThaumarchaeota account for up to 40% of totalmicrobes in meso- and bathypelagic deep oceanwaters (Karner et al., 2001). In analogy, we presentthe first quantitative data for the dominance of MCGcrenarchaeota in anoxic marine sediments. We useda combination of three methods that are based ondifferent target molecules, that is, DNA and RNA, toprovide robust environmental data for MCG
independent of their individual methodologicalconstrains. All methods pointed to a higher relativeabundance of MCG to total microbes in sediments atsites where sulfate penetrates at least 10 cm.
Cold seeps with high methane fluxes and highmicrobial respiration rates are characterized by lowsulfate penetration depths. Among the cold seepsites with shallow sulfate depletion investigatedhere, the four sites from Hydrate Ridge and site 156from the Gulf of Mexico showed the lowest MCGabundance concurrent with extremely high rates ofAOM and sulfate reduction (Boetius et al., 2000;Treude et al., 2003; Orcutt et al., 2010). Anexception were 4.6 m-deep sediments from sites371 and 372 at HMMV which showed a high MCGabundance together with the lowest microbialactivity due to high fluid flow rates repressingsulfate penetration (DeBeer et al., 2006; Niemannet al., 2006).
Sediments from ‘low-energy’ environments orfrom cold seeps with low methane fluxes and lowmicrobial respiration rates are characterized by deepsulfate penetration. In all non-seep, low-energyhabitats, MCG made up a high proportion of MCGrelative to total prokaryotes, ranging from 12% at
Figure 6 Slot blot-based determination of the ratio archaea:bacteria 16S rRNA in selected surface and subsurface sediments. The sum ofdetected archaeal and bacterial 16S rRNA was set as 100% of prokaryotic rRNA. The archaeal fraction is further resolved in the columnthat shows the proportion of MCG and other, yet unidentified archaea.
MCG crenarchaeota in marine sedimentsK Kubo et al
1961
The ISME Journal
Janssand to 45% at Peru Margin ODP 1229D or evenup to 100% at White Oak River. This finding wasindependent of water depths, temperature or totalcarbon contents (organic-poor ODP sites 1225 and1231 vs organic-rich ODP sites 1227 and 1229).Therefore, it appears that MCG dominate thearchaeal population in low activity subsurfacesediments. At cold seeps with deep sulfate penetra-tion such as Hydrate Ridge site ODP1245 andODP1250 as well as Gulf of Mexico site 87, relativeabundance of MCG was comparably high (15–74%of archaea) as for non-seep sediments with deepsulfate penetration. An exception was HMMV seepsite 336 that showed the lowest relative percentagesof MCG of all CARD-FISH counts (3% of archaea).However, the deep sulfate penetration depth at thisspecial site is not the result of low microbialrespiration rates, but instead is driven by activepumping of sulfate into the core by Siboglinidaetubeworms (DeBeer et al., 2006).
No support for an association of MCG with the methanecycleData obtained in this study do not support thehypothesis that MCG could be anaerobic methano-trophs (Biddle et al., 2006). MCG distribution is notproportional to any geochemical gradients, andspecifically, does not co-vary with energy availabil-ity from methane and sulfate. We did not findevidence for changes of MCG activity with depthbased on rRNA content per MCG cell (range 1–117 fgper cell, calculated based on rRNA slot blot andCARD-FISH). When the White Oak River wasstudied with a finer depth resolution, this trendwas upheld. Similar to many diffusion-drivenmarine sediments, those of the White Oak Riverestuary are stratified with respect to dominantmicrobial electron acceptor. Thus, if MCG crenarch-aeota depend on a known terminal electron acceptoreither directly or indirectly through microbialsyntrophy, MCG presence, abundance or activitywould be expected to change downcore. However,we found no evidence for such a coupling withdepth. In contrast, sediments from Hydrate Ridgesite 19-2 had a peak in MCG abundance (qPCR) andactivity (slot blot) coinciding with the bottom of thesulfate reduction zone where highest methaneoxidizing activity is presumed to occur (Knittelet al., 2003). Nevertheless, in agreement with WhiteOak River sediments, the percent MCG of totalarchaea had no depth trend. Further support for alack of vertical zonation of MCG came from the lackof MCG phylogenetic composition changes withdepth in White Oak River sediments. Secondly, acomparison of MCG cell numbers in seep (forexample, Hydrate Ridge, Gulf of Mexico) vs non-seep (for example, North Sea, Peru, White Oak Riverestuary) sediments did not show remarkable differ-ences, whereas numbers of methanotrophs changedramatically across all the sites investigated (see
Knittel and Boetius, 2009). The lack of verticalzonation together with similar cell numbers of MCGat seeps and non-seep sediments suggest that MCGare anaerobes, as they maintain a high abundancewell-below oxygen penetration, but do not gainenergy from AOM and sulfate is likely not anelectron acceptor. This, together with a widespreaddistribution of MCG, could indicate that they obtaincarbon and energy from substrate pools and energysources that vary little with sediment depth andbetween habitats. Options for such metabolismsinclude the slow fermentative degradation of differ-ent types of organic matter. MCG are likely to accesssubstrates that are physically or chemically recalci-trant. This conclusion is in agreement with findingsfrom previous literature (Biddle et al., 2006; Lippet al., 2008; Takano et al., 2010; Webster et al., 2010;Lliros et al., 2011).
Conclusions
The high diversity and high abundance of MCGcrenarchaeota in seep and non-seep sedimentssuggest that they are globally important componentsof the anaerobic sedimentary microbial community.However, the question of what carbon and energysources sustain MCG is still open. The lack of abruptchanges in MCG abundance or phylogenetic compo-sition at the downcore transition from zones withsulfate reduction/methane oxidation to those withmethane production as well as the lack of differencein MCG cell numbers at seeps vs non-seeps suggeststhat the MCG community is not active in methane orsulfur cycling. This is supported by a muchhigher relative abundance of MCG compared withother prokaryotes in low-energy environments. It is,therefore, likely that MCG are anaerobic hetero-trophs and either access a wide variety of fermenta-tive substrates or are linked to degradation ofrefractory organic matter, potentially using otherelectron acceptors than sulfate. This possible con-tribution to post-depositional carbon remineraliza-tion makes MCG a promising target for futureculturing and ecophysiological research.
Acknowledgements
We greatly acknowledge Antje Boetius for fruitful discus-sions and for providing samples from expeditions with theresearch vessels SONNE, POLARSTERN, POURQUOIPAS?, Tina Treude for samples from POSEIDON andKai-Uwe Hinrichs and Katharina Kohls for sediments fromODP sites and hypersaline mats, respectively. Sampleswere taken in the framework of the GEOTECHNOLOGIENprograms MUMM I and II (grants 03G0554A and03G0608A) funded by the German Ministry of Educationand Research (BMBF) and the German Research Founda-tion, by the EU 6th FP HERMES, and by the Ocean DrillingProgram (Leg 201 and 204) funded by the National ScienceFoundation (NSF) and participating countries under theJoint Oceanographic Institutions. Dirk Rickert is acknowl-edged for sulfate measurements in Hydrate Ridge
MCG crenarchaeota in marine sedimentsK Kubo et al
1962
The ISME Journal
sediments. Howard Mendlovitz, Daniel Albert, JohnBiddle, Andrew Steen, and Luke McKay helped with coreacquisition and sectioning. Niculina Musat and BarbaraMacGregor are acknowledged for helpful discussionsabout RNA extraction and slot blot hybridization. Finan-cial support for K Kubo came from the German AcademicExchange Service (DAAD), for KGL from the US Environ-mental Protection Agency Star Fellowship, the DanishNational Research Foundation. JFB was supported by aNASA NPP Fellowship administered by ORAU; and ATwas supported from NOAA-NIUST and the NASA Astro-biology Institute for 16S clone library sequencing, and theKeck Foundation for V6-tag sequencing. Further supportwas provided by the Max Planck Society.
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